Bottom Line:
All immortalized mesothelial clones consistently grow in DMEM supplemented with fetal bovine serum.Cells can be passaged for more than 40 times without any signs of morphological changes or a decrease in proliferation rate.RN5 cells are highly tumorigenic.

ABSTRACTMesothelial cells are susceptible to asbestos fiber-induced cytotoxicity and on longer time scales to transformation; the resulting mesothelioma is a highly aggressive neoplasm that is considered as incurable at the present time Zucali et al. (Cancer Treatment Reviews 37:543-558, 2011). Only few murine cell culture models of immortalized mesothelial cells and mesothelioma cell lines exist to date. We generated SV40-immortalized cell lines derived from wild-type (WT) and neurofibromatosis 2 (merlin) heterozygote (Nf2+/-) mice, both on a commonly used genetic background, C57Bl/6J. All immortalized mesothelial clones consistently grow in DMEM supplemented with fetal bovine serum. Cells can be passaged for more than 40 times without any signs of morphological changes or a decrease in proliferation rate. The tumor suppressor gene NF2 is one of the most frequently mutated genes in human mesothelioma, but its detailed function is still unknown. Thus, these genotypically distinct cell lines likely relevant for malignant mesothelioma formation are expected to serve as useful in vitro models, in particular to compare with in vivo studies in mice of the same genotype. Furthermore, we generated a novel murine mesothelioma cell line RN5 originating from an Nf2+/- mouse subjected to repeated crocidolite exposure. RN5 cells are highly tumorigenic.

Mentions:
Primary mesothelial cells from WT mice, immortalized cells iMeso-WT1, iMeso-NF3, and mesothelioma cells from the lines AK7 and RN5, all derived from a C57Bl/6J background, were injected (4 × 105 cells) subcutaneously into the right flank of C57Bl/6J mice. Tumor formation was observed in all four mice injected with RN5 cells (Fig. 6a). No macroscopically evident (palpable) tumors were observed in mice injected with primary mesothelial cells of WT origin or with immortalized cells (iMeso). No tumors developed after injection of the selected number of AK7 cells; a significantly higher number (>2 × 106 cells) has been reported to be required in order to induce macroscopic tumors in this experimental model (Cordier Kellerman et al.2003). The tumor tissue originating from RN5 cells isolated 59 d after injection was white in color and rather solid at palpation. On histological sections subjected to a Goldner staining, a fibrous stroma was evident (Fig. 6c). Histological examination of tumors (Fig. 6b) derived from RN5 cells isolated 59 d after injection revealed a proliferation of atypical cells with a sarcomatoid morphology. The tumor was mainly confined to the subcutaneous tissue with infiltration of small clusters as well as individual cells into the dermis. Immunohistochemically, the tumor cells were positive for vimentin with strong coexpression of pancytokeratin in part of the cell population (Fig. 6d, e). No expression was seen for WT1 and calretinin (data not shown). The morphology combined with staining for vimentin and pancytokeratin is consistent with a sarcomatoid mesothelioma.Figure 6.

Mentions:
Primary mesothelial cells from WT mice, immortalized cells iMeso-WT1, iMeso-NF3, and mesothelioma cells from the lines AK7 and RN5, all derived from a C57Bl/6J background, were injected (4 × 105 cells) subcutaneously into the right flank of C57Bl/6J mice. Tumor formation was observed in all four mice injected with RN5 cells (Fig. 6a). No macroscopically evident (palpable) tumors were observed in mice injected with primary mesothelial cells of WT origin or with immortalized cells (iMeso). No tumors developed after injection of the selected number of AK7 cells; a significantly higher number (>2 × 106 cells) has been reported to be required in order to induce macroscopic tumors in this experimental model (Cordier Kellerman et al.2003). The tumor tissue originating from RN5 cells isolated 59 d after injection was white in color and rather solid at palpation. On histological sections subjected to a Goldner staining, a fibrous stroma was evident (Fig. 6c). Histological examination of tumors (Fig. 6b) derived from RN5 cells isolated 59 d after injection revealed a proliferation of atypical cells with a sarcomatoid morphology. The tumor was mainly confined to the subcutaneous tissue with infiltration of small clusters as well as individual cells into the dermis. Immunohistochemically, the tumor cells were positive for vimentin with strong coexpression of pancytokeratin in part of the cell population (Fig. 6d, e). No expression was seen for WT1 and calretinin (data not shown). The morphology combined with staining for vimentin and pancytokeratin is consistent with a sarcomatoid mesothelioma.Figure 6.

Bottom Line:
All immortalized mesothelial clones consistently grow in DMEM supplemented with fetal bovine serum.Cells can be passaged for more than 40 times without any signs of morphological changes or a decrease in proliferation rate.RN5 cells are highly tumorigenic.

ABSTRACTMesothelial cells are susceptible to asbestos fiber-induced cytotoxicity and on longer time scales to transformation; the resulting mesothelioma is a highly aggressive neoplasm that is considered as incurable at the present time Zucali et al. (Cancer Treatment Reviews 37:543-558, 2011). Only few murine cell culture models of immortalized mesothelial cells and mesothelioma cell lines exist to date. We generated SV40-immortalized cell lines derived from wild-type (WT) and neurofibromatosis 2 (merlin) heterozygote (Nf2+/-) mice, both on a commonly used genetic background, C57Bl/6J. All immortalized mesothelial clones consistently grow in DMEM supplemented with fetal bovine serum. Cells can be passaged for more than 40 times without any signs of morphological changes or a decrease in proliferation rate. The tumor suppressor gene NF2 is one of the most frequently mutated genes in human mesothelioma, but its detailed function is still unknown. Thus, these genotypically distinct cell lines likely relevant for malignant mesothelioma formation are expected to serve as useful in vitro models, in particular to compare with in vivo studies in mice of the same genotype. Furthermore, we generated a novel murine mesothelioma cell line RN5 originating from an Nf2+/- mouse subjected to repeated crocidolite exposure. RN5 cells are highly tumorigenic.